The Study On The Mechanisms Of Anti-neoplastic Effect Of Metformin In Thyroid Cancer

Objective:1. To assess the effect of metformin on anti-proliferation and apoptosis of human thyroid carcinoma cells, and demonstrate the anti-neoplastic effect of metformin in thyroid cancer.2. To investigate the excessive activation of epithelial-mesenchymal transition (EMT) in thyroid carcinoma cells, and study the inhibitive effect of metformin on invasive and metastatic abilities of such cells.3. To explore the underlying mechanisms of metformin on growth, invasion and metastasis of human thyroid carcinoma cells.Methods:1. Human anaplastic thyroid cancer cell line SW1736, follicular thyroid cancer cell line HFO and primary thyrocytes were treated with different concentrations of metformin. MTT assay was used to detect proliferation viability of different cells. Cell cycle progression and apoptosis were analyzed by flow cytometery.2. Agarose drop explant assay, scratch wound assay, transwell invasion and migration assay were arranged to compare the invasion and metastasis ability of SW1736 and HFO cells in different metformin treated groups.3. After cultured with metformin, quantitative real-time PCR was performed to measure the mRNA expression of vimentin、E-cadherin、Snail1 and Slug in SW1736 and HFO cells.4. Expressions of vimentin、AMPK、pAMPK、mTOR and pmTOR proteins were analysed by western blot in SW1736 and HFO cells which were treated with different doses of metformin.Result:1. Metformin significantly inhibited the growth of SW1736 and HFO cells in a time-and dose-dependent manner, with a slightly inhibition effect on human primary thyrocytes in vitro.2. Metformin decreased cell invasive and metastatic abilities in SW1736 and HFO cells, which were analysed by agarose drop explant assay, scratch wound assay, transwell invasion and migration assay.3. The results of flow cytometry showed that metformin inhibited cell cycle progression in SW1736 and HFO cells. Furthermore, metformin also enhanced apoptosis of SW1736 cells in a dose-dependent manner. Compared with the control group, addition of 20 mmol/L metformin for 48 h increased the percentage of apoptotic cells from 8.3% to 13.3% in SW1736 cells.However, in human primary thyrocytes, no significant effect was observed in cell cycle progression and cell cycle progression.4. Expression of vimentin was observed in SW1736 and HFO cells, and showed a significant down-regulation after cultured with metformin. Up expression of E-cadherin was observed in SW1736 cells after treated with metformin. Addition of 20 mmol/L metformin for 48 h induced E-cadherin gene expression 7.81±3.00 fold-change than the control group.5. Compared with the control group, Slug gene expression showed a significant down-regulation both in SW1736 and HFO cells. After treated with 20 mmol/L metformin for 48 h, analysis of Snaill gene expression showed a significant down-regulation in SW1736 cells(0.13±0.00 fold-change, p<0.01). These results suggested that metformin induced E-cadherin expression by inhibitng Snail1 and Slug gene expression, then suppressed EMT process and decreased migration and invasion ability in human thyroid carcinoma cells.6. Compared with the control group, metformin significantly actived phosphorylation of AMPK and reduced phosphorylation of mTOR. Metformin exerted the effects by activating AMPK signaling and inhibiting mTOR signal pathway.Conclusion:1. Metformin can inhibit proliferation and cell cycle progression and induce apoptosis of thyroid carcinoma cell lines.2. Metformin can decrease the migration and invasion ability by suppressing EMT process in human thyroid carcinoma.3. Metformin can inhibit the over-activation of mTOR signal pathway by directly activating AMPK, thus block the EMT process in thyroid cancer. In conclusion, metformin may be a potential therapeutic agent and improve the effectiveness of human thyroid carcinoma therapy.